Pichia Classic: Choosing the right strain and vector system:
There are several approaches to choosing the best Pichia Classic strain and vector system for your protein of interest. If you do not know which strain or vector to choose as a first time user, then either the Original Pichia Expression Kit or the EasySelect Pichia Expression Kit is a good option as each kit provides a variety of strains and vectors to test for the one which works best for your particular protein. The kits also include all the required reagents to get started.
If you are an experienced Pichia user or have specific preferences based on your protein of interest, then there are several options. First, determine if you prefer selection of successful transformation using an antibiotic (Zeocin or Blasticidin) or using a HIS– strain and histidine-deficient medium. Pichia Classic strain GS115 has a mutation in the HIS4 gene (HIS4–) and requires the use of vectors containing the HIS4 gene (pPIC vectors) and successfully transformed clones will grow in medium lacking histidine. The Pichia Classic X-33 strain retains a functional HIS4 gene (HIS4+) and would be used with other selectable markers like Zeocin (pPICZ vectors) or Blasticidin (pPIC6 vectors). Another consideration is to use methanol induction under the AOX1 promoter or to constitutively express the protein under the GAP promoter. A Pichia Classic strain with a “mut+” designation means that the strain has operable AOX1 and AOX2 genes (GS115, X-33, SMD1168) and is fast growing upon methanol induction. A Pichia Classic strain with a “muts” designation has a knocked-out AOX1 gene and is slow growing upon methanol induction (KM71, KM71H). Finally, if you believe that your protein will be susceptible to protease degradation, then there are two protease deficient strains, SMD1168 (HIS4–) and SMD1168H (HIS4+), both lacking protease A activity.
With regard to selecting a vector system, you should consider several factors:
- Promoter induction: Several different promoters can be used to drive protein production. One should start with the pPICZ vectors that contain the AOX1 promoter since methanol induction is the driver of high protein yields in Pichia Classic. If you also want to try the constitutive GAP promoter, it is available in the pGAPz vectors. Finally, in a limited number of cases, a formaldyhyde promoter is also available (pFLD1 kit).
- Selection of transformants: The use of antibiotics (Zeocin or Blastocidin) versus histidine-auxotrophy is readily available for selection of transformants.
- Intracellular production vs. extracellular secretion: One big advantage of Pichia is the ability to secrete the target protein into the medium (vector should contain the alpha mating factor, vector has an ending “a” designation). Alternatively, the protein can be produced intracellularly.
- Single-copy vs. Multi-copy gene insertions: Multi-copy gene insertions can lead to high protein expression. If you require multi-copy insertion of your target gene, you can use the Multi-Copy Pichia Expression Kit.
Life Technologies SKU
|K1710-01||Original Pichia Expression Kit||Contains the strains, GS115 and KM71, plus 4 vectors and reagents. Geared for histidine selection.|
|K1740-01||EasySelect Pichia Expression Kit||Contains the strains GS115, X-33 and KM71, along with the pPICZ vectors and reagents.|
|K1780-01||Multi-Copy Pichia Expression Kit||Contains GS115, KM71 and the pPIC9K vectors for multi-copy gene insertion.|
|C175-00||SMD1168||Protease A deficient, HIS4-|
|C184-00||SMD1168H||Protease A deficient, HIS4+|
|C182-00||KM71H||HIS4+, Mut-s, aox1-|
|V190-20/V195-20||pPICZ and pPICZα||For use with all strains and zeocin selection. AOX1 promoter. A, B, C designations refer to the cloning site reading frame|
|V200-20/V205-20||pGAPZ/pGAPZα||GAP promoter, same as pPICZ|
|V210-20/V215-20||pPIC6/pPIC6α||AOX1 promoter and blasticidin resistance gene.|
|V173-20/V175-20/V180-20||pPIC3.5K/pPIC9K/pAO815||For multicopy gene insertion|
|V230-20||pFLD1 vector kit||FLD promoter|
Access Life Technologies Pichia Ordering page and additional information here Life Technologies
Download the Life Technologies Product Manual for Pichia Classic Kits Original Pichia Kit Manual